Maéno M et al. (1994), A truncated bone morphogenetic protein 4 recept...

XB-ART-20662

Proc Natl Acad Sci U S A 1994 Oct 25;9122:10260-4.

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A truncated bone morphogenetic protein 4 receptor alters the fate of ventral mesoderm to dorsal mesoderm: roles of animal pole tissue in the development of ventral mesoderm.

Maéno M , Ong RC , Suzuki A , Ueno N , Kung HF .

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The biological effects of endogenous bone morphogenetic protein 4 (BMP-4), a member of the transforming growth factor beta family, on embryonic development of Xenopus laevis were investigated by using a functionally defective mutant of the BMP-4 receptor (delta mTFR11), which blocks the BMP signaling pathway. Injection of delta mTFR11 RNA into either the animal pole area or ventral marginal cells at the two-cell stage induced a dorsal phenotype in the explant of ventral mesoderm with animal pole tissue from stage 10+ embryo, even though the normal fate of this explant is a "mesenchymal ball" containing blood cells. These explants with the dorsal phenotype contained muscle, neural tissue, eye capsule, and cement gland. Northern blot analysis showed an increase of cardiac alpha-actin mRNA and a decrease of T alpha-globin mRNA expression, providing further evidence of a conversion from ventral to dorsal phenotype. Although injection of delta mTFR11 RNA did not induce mesoderm in an animal cap culture, the same tissue injected with delta mTFR11 RNA can alter the differentiation fate of uninjected ventral mesodermal explant from ventral to dorsal type, suggesting specific interaction of animal pole tissue and prospective ventral mesoderm in vivo.

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Species referenced: Xenopus laevis
Genes referenced: acta2 actl6a bmp4 bmpr1a bmpr1b hba1 hba3

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	FIG. 1. Morphological observation of the 3-day explants of ventral mesoderm (VM) with animal pole (AP) tissue injected with H20 (A) or AmTFR11 RNA (B-D). In A-C, H20 (A) or 5 ng of A&mTFR11 RNA (B and C) was injected into the animal pole area at the two-cell stage, and the resulting animal pole tissue at stage 10+ was excised together with ventral mesoderm (B) or combined with ventral mesoderm from a different embryo (C). In D, 5 ng of AmTFR11 RNA was injected into ventral mesoderm, and the resulting ventral mesoderm at stage 10+ was excised together with animal pole tissue. Injection of AmTFR11 RNA induced a tight structure, similar to a typical dorsal mesoderm explant. Note the obvious eye-like capsule and cement gland that were found in the explants injected with AmTFR11 RNA into the animal pole area (B and C).
	FIG. 2. Histological observation of the 3-day explants of ventral mesoderm with animal pole tissue after injection of AmTFR11 RNA (A-C) or H20 (D). Five nanograms of AmTFR11 RNA was injected into the animal pole area (A and B) at the two-cell stage or into the ventral marginal zone (C) at the four-cell stage, and the resulting tissue at stage 10+ was excised with ventral mesoderm (A and B) or with animal pole tissue (C), respectively. Neural tissues (n) and muscle (m) were differentiated in the explants injected with AmTFR11 (A-C). However, notochord (nc) was only seen in the explants injected with AmTFR11 RNA into ventral mesoderm (C). In contrast, a typical ventral mesoderm structure containing blood cells (b) was developed in the explants injected with H20 (D, control). A and B were different sections of the embryos. (E and F) Sections of explant in a lineage-tracing experiment using P-galactosidase as a marker. One-half nanogram of capped RNA coding P-galactosidase was injected into the ventral marginal zone at the four-cell stage, and the resulting ventral mesoderm at stage 10+ was combined with animal pole tissue that was previously injected with 5 ng of AmTFR11 RNA (E) or was combined with animal pole tissue without injection of AmTFR11 (F). Explants were cultured for 3 days, fixed, processed for 5-bromo-4chloro-3-indolyl ,3-D-galactoside staining, and sectioned. Note that ventral mesoderm-derived cells, which express ,-galactosidase, differentiated to muscle tissue by the injection with AmTFR11 RNA into the animal pole area (E). In the control without injection of AmTFR11 RNA, these cells differentiated to blood cells and mesenchymal cells (F). (Bars in A and E = 50 uan; same magnification in A-D and E and F, respectively.)
	FIG. 3. Morphology (A) and Northern blot analysis (B) of the explants injected with increasing doses of AmTFR11 RNA. Expression of cardiac a-actin, Ta-globin, and EF-la mRNAs was determined by Northern blot analysis. The embryo was injected with AmTFR11 RNA into the animal pole area at the two-cell stage, and the resulting tissue at stage 10+ was excised together with ventral mesoderm and cultured for 3 days. Various concentrations of AmTFR11 were used: sample 1, H20 injection; sample 2, 0.04 ng of RNA; sample 3, 0.2 ng of RNA; sample 4, 1 ng of RNA; sample 5, 5 ng of RNA; sample 6, dorsal mesoderm and animal pole tissue cultured as a control.
	FIG. 4. Effect of activin and AmTFR11 RNA injection on the expression of cardiac a-actin and Ta-globin mRNA expression. Northern blot analysis was performed to quantitate the expression of Ta globin, cardiac a-actin, and EF-la mRNAs. In lanes 1-4, RNA was injected into the animal pole area at the two-cell stage, and the resulting tissue at stage 10+ was excised together with ventral mesoderm and cultured for 3 days. Lane 1, no injection; lane 2, 5 ng of AmTFR11 RNA; lane 3, 0.5 ng of activin (8A RNA; lane 4, dorsal mesoderm with animal pole tissue cultured as a control. In lanes 5-8, RNA was injected into the animal pole area at the two-cell stage, and the animal cap at stage 7 was cultured for 3 days. Lane 5, no injection; lane 6,0.5 ng of activin PA RNA; lane 7,5 ng ofAmTFR11 RNA; lane 8, 5 ng ofXenopus BMP-4 RNA. Dorsal mesoderm with animal pole tissue (lane 9) or ventral mesoderm with animal pole tissue (lane 10) was cultured for 3 days as a control. Note that activin and AmTFR11 RNA can dorsalize the explants of ventral mesoderm with animal pole tissue (lanes 2 and 3), but only activin can induce mesoderm in the animal cap explant (lane 6).
	FIG. 5. Hypothetical roles of BMP on the ventralzing and dorsalizing activities. In the normal embryo without AmTFR11 RNA injection (A), endogenous BMP signaling stimulates and enhances ventralizing activity (VA) and ventralizes (V) the adjacent prospective ventral mesoderm (VM). At the same time, the dorsalizing activity (DA) is inhibited by BMP signal(s). In the embryo injected with AmTFR11 RNA (B), the disruption of BMP signaling in the animal pole region (AP) relieves the inhibition of dorsalizing activity of BMP and turns on the dorsalizing activity and dorsalizes (D) the adjacent prospective ventral mesoderm. Plus represents stimulatory activity and minus represents inhibitory activity. A solid line indicates that the signals are transmitted, and a dotted line indicates that the signals are inhibited or abrogated.

References [+] :

Amaya, Expression of a dominant negative mutant of the FGF receptor disrupts mesoderm formation in Xenopus embryos. 1991, Pubmed, Xenbase